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Abstract

Background

Multiple pregnancies are an important complication of assisted reproductive technology (ART). The present study aims to indentify the risk factors for multiple
pregnancies independent of the number of transferred embryos.

Materials and Methods

This retrospective study reviewed the medical records of patients who underwent intracytoplasmic sperm injection (ICSI) cycles in Royan Institute
between October 2011 and January 2012. We entered 12 factors that affected the number
of gestational sacs into the poisson regression (PR) model. Factors were obtained from
two study populations-cycles with double embryo transfer (DET) and cycles that transferred
three embryos (TET). We sought to determine the factors that influenced the number of gestational sacs.
These factors were entered into multivariable logistic regression
(MLR) to identify risk factors for multiple pregnancies.

Results

A total of 1000 patients referred to Royan Institute for ART during the study period.
We included 606 eligible patients in this study. PR analysis demonstrated that the quality of
transferred embryos and woman’s age had a significant effect on the number of observed sacs
in patients who underwent ICSI with DET. There was no significant predictive variable for
multiple pregnancies according to MLR analysis. Our findings demonstrated that both regression
models (PR and MLR) had the same outputs. A significant relation existed between age
and fertilization rate with multiple pregnancies in patients who underwent ICSI with TET.

Conclusion

Single embryo transfer (SET) should be considered with the remaining embryos
cryopreserved to prevent multiple pregnancies in women younger than 35 years of age who
undergo ICSI cycles with high fertilization rates and good or excellent quality embryos. However,
further prospective studies are necessary to evaluate whether SET in women with these
risk factors can significantly decrease multiple pregnancies and improve cycle outcomes.

Introduction

At present, the use of assisted reproductive
technology (ART) is expanding worldwide. A related
challenge after ovarian hyperstimulation is
multiple gestation and the health of children born
by ART. Recently, the rate of multiple pregnancies
has dramatically increased due to the widespread
use of ART (1). Multiple pregnancies are
associated with increased risk of maternal and
fetal complications (2). The ideal of infertility
therapy is to achieve one healthy baby at a time
(3). Despite the attempts to limit the incidence of
multiple pregnancies after ART by elective single
embryo transfer (SET), the average in vitro fertilization
(IVF) treatment includes transfer of two,
three or sometimes more embryos into the uterus.
SET, as a clinical practice, has not been executed
in some countries.

Previous studies evaluated embryo and cyclespecific
parameters associated with twin pregnancies
after double embryo transfer (DET)
(4-8). Niu et al. (4) found that four factors-the
first treatment cycle, good ovarian response,
higher number of top-quality embryos, and development
stage score of the second-best embryo
transferred had an independent association with
twin pregnancies after DET. Xu et al. (5) reported
that women’s age and the number of highquality
embryos transferred were risk factors
associated with twin pregnancies after IVF with
DET. Groeneveld et al. (6) demonstrated that the
height of the women and the number of oocytes
retrieved were associated with an increased risk
of twins after DET.

In a recent study, Kim et al. (8) concluded that
younger age, higher body weight, and better
quality of transferred embryos showed an association
with increased chance for twin pregnancies
after DET at cleavage stage. Identification
of risk factors for multiple pregnancies can enable
medical personnel to provide counseling and
information to couples at high risk for multiple
pregnancies and suggest SET in their infertility
treatment program. The aim of present study is
to evaluate the factors that affect the occurrence
of multiple pregnancies after transfer of two or
three embryos in intracytoplasmic sperm injection
(ICSI) by two different regression analyses
(poisson and Logistic).

Materials and Methods

This retrospective cohort study reviewed the
records of patients referred to Royan Institute between
October 2011 and January 2012. The Institutional
Scientific Board of Royan Institute approved
this study. Admitted patients gave written
consent that stated which the treatment information
would be used for scientific purposes without
mentioning names or personal details.

The data related to infertile couples who underwent
ICSI cycles and included: demographic
information, medical records, and cycle characteristics.
Excluded from the study were: women
older than 40 years, uterine factor infertility
(myoma, polyps, and congenital malformations),
recurrent miscarriages, and stages II or III endometriosis.
Cycles with donor oocytes or embryos,
preimplantation genetics diagnosis, and blastocyst
embryo transfer were also excluded. In this
study we reviewed all ICSI cycles that had complete
data. Embryo quality was recorded based on
the fragmentation degree and regularity of blastomeres
on day 3 after fertilization, as follows
(9): excellent: 6-8 equal sized blastomeres with
≤10% frag mentation; good: 6-8 equal or unequal
sized blastomeres with 10-20% fragmentation;
and fair: uneven sized and few blastomeres with
>20% fragmentation. We calculated the fertilization
rate according to the number of fertilized oocytes
per number of microinjected MII oocytes.
Multiple pregnancies were considered as the observation
of more than one gestational sac with
heart beats by vaginal ultrasound evaluation six
weeks after embryo transfer.

Statistical analysis

The findings were described as mean ± SD for
quantitative variables and number (%) for qualitative
variables. We compared the study population
characteristics according to the number of
embryos transferred, either DET or triple embryo
transfer (TET) according to the student’s t test
and chi-square test for quantitative and qualitative
variables, respectively. To investigate the
relationship between factors and gestational sac,
we used the statistical software STATA 11 program
and the logistic and poisson regression (PR)
models. On the basis of previous studies (5, 8, 10,
11), the possible variables that affected the number of gestational sacs included: women’s age,
body mass index (BMI), infertility type, infertility
cause and duration, acquired uterine anomaly,
endometrial thickness on the day of the human
chorionic gonadotropin (hCG) injection, type of
stimulation protocol, present cycle type, ovarian
response type, total number of gonadotropin ampules,
total number of retrieved oocytes, number
of MII oocytes, fertilization rate, and quality of
transferred embryos were listed in the PR model
for both study populations (DET and TET). Multiple
logistic regression (LR) model was used to
determine significant variables related to multiple
pregnancies. We considered P<0.05 to be statistically
significant.

Results

A total of 1000 patients referred to Royan Institute
for ART during the study period. We included 606
patients in this study. There were 336 patients with
DET and 270 patients with TET. Table 1 lists the
demographic and medical characteristics of patients
according to group (DET or TET). The women’s
mean age, duration of infertility, and number of
previous ART cycles in the TET group were greater
than those in the DET group (P<0.001). The two
groups showed no significant differences in terms
of BMI, infertility type, endometrial thickness, and
quality of transferred embryos. The DET group had
a higher mean number of retrieved and MII oocytes.
Despite a higher fertilization rate in the TET group,
we observed clinical pregnancy rate between the
two groups (P=0.1).

The distribution of the frequency of the numberof
sacs in the DET and TET groups were shown in
Table 2. The results showed that the mean of the
observed gestational sac in the TET group (0.64
± 0.88) was greater than the DET group (0.48 ±
0.72, P=0.01). We calculated incidence rate ratios
(IRRs) for explanatory variables by the PR models
to identify the related variables to the number of
observed gestational sacs after the ICSI cycles. If
an IRR is greater than 1, the incidence rate (IR,
sac no.) increases as x (explanatory variable) increases.
If an IRR is less than 1, the IR decreases
as x increases.

The quality of the transferred embryos and woman's
age significantly impacted the number of observed
sacs in the DET group (Table 3,). It means that
the IR of multiple sac in excellent grade embroyos
group was 9 times and meanwhile in good grade
embroyos group was 6 times respect to fair grade
embryos as refrence group. The results showed that
the IRR for women’s age was 0.96 (95% CI: 0.93-
0.99). In other words, one year increases in female
age showed an IR of the observed gestational sac
that decreased 4%. The other explanatory variables
did not impact the number of sacs.

In the TET group, women's age, type of infertility,
and fertilization rate significantly impacted the
number of sacs (Table 4,). The IRR for maternal
age was 0.94 (95% CI: 0.90-0.97) and IRR for the
fertilization rate was 6.02 (95% CI: 2.96-12.22).
The one year increase in female age showed an IR
of the observed gestational sac that decreased 6%.
The IR of the observed gestational sac increases 6
times when one unit increases in the fertilization
rate. The results demonstrated that the IR of the
observed gestational sac in patients with secondary
infertility decreased 42% with respect to those
with primary infertility (Table 4,).

All the possible affecting variables that included
women’s age, BMI, infertility type, infertility cause
and duration, acquired uterine anomaly, endometrial
thickness on the day of the hCG injection, type
of stimulation protocol, present cycle type, ovarian
response type, total number of gonadotropin ampules,
total number of retrieved oocytes, number of
MII oocytes, fertilization rate, and quality of transferred
embryos were entered in the multiple logestic
regression (MLR) model. The result showed that
women's age, duration of infertility, and number of
transferred embryos were the most important variables
related to multiple pregnancies in this population
(Table 5,). Therefore, we repeated the MLR
analysis in the two separate populations (DET and
TET). The results showed that none of the variables
in the model had a significant association with multiple
pregnancies in the DET group. Multiple LR
showed that in the TET group, women's age and
fertilization rate were significantly related variables
(Table 6). Women with increased age had a 20% reduction
in the odds for multiple pregnancies. When
the fertilization rate increased one unit, the odds for
a multiple pregnancy increased 19.7 times.

Discussion

In present study, on the basis of the PR model, we
found that the quality of transferred embryos and
women’s age had a significant effect on the number
of observed sacs in patients who underwent ICSI
cycles with DET. However, based on the multiple
LR model, we found no significant predictive
variable for multiple pregnancy in these patients.
Previous studies reported that maternal age (5, 8),
body composition (6, 8), good ovarian response,
cycle number, and the number of retrieved oocytes
(4) had a relationship to multiple pregnancies after
DET. The current study results showed no impact
by body composition, cycle number, number of retrieved
oocytes, and type of ovarian response. In
agreement with previous studies (5, 8), we found
that the quality of transferred embryos significantly
influenced the IR of sac numbers. Kaser et al.
(7) stated that there were six risk factor for twin
live births after cryopreserved cleavage stage DET
cycles: age <35 years, resumption of mitosis, 7-8
viable cells in the non-lead embryo, transfer of a
lead embryo with ≥7 cells and a total of ≥14 viable
cells.

Groeneveld et al. (6), in a large nationwide Dutch
cohort, demonstrated that tall stature and increased
number of retrieved oocytes independently increased
the chance for dizygotic twins after IVF
with DET. In contrast to this study, we did not find
any relationship between multiple pregnancies and
BMI or number of retrieved oocytes in ICSI cycles
with DET.

In agreement with Niu et al. (4), we found that
excellent and good quality transferred embryos
independently increased the chances of multiple
implantation after ICSI with DET. They suggested
that it was advisable to perform SET when patients
had high risk factors for twin pregnancies
that included initial IVF-ET treatment, good or
high ovarian response, more number of top-quality
embryos, and development stage score of the
second best embryo transferred. Fauque et al. (12)
concluded that not only the implantation and pregnancy
rates, but also the live birth rate depended
on embryo quality.

On the basis of our knowledge, the present study
was the first to evaluate risk factors for multiple
pregnancies in patients who underwent ICSI with
TET. Our findings demonstrated that both regression
models (poisson and logistic) had the same
outputs. In both models, women’s age and fertilization
rate showed significant relationships with
multiple pregnancies in these patients. It could be
taken as a measure of oocyte quality, which has
been shown to be associated with increased implantation
potential. A higher rate of fertilization
would likely result in a higher number of MII oocytes
and, consequently, a higher chance of good
quality embryos would be associated with higher
chances for multiple pregnancies. In agreement
with previous studies we found a negative correlation
between female age and multiple pregnancies
in ICSI cycles with TET. Younger women had increased
implantation potential and multiple pregnancies
(10, 11).

In the present study, we have sought to evaluate
the risk factors of multiple pregnancies after transfer
of two or three embryos in ICSI cycles. If the
risk factors of multiple pregnancies could be identified,
we can give proper counseling to couples at
risk for multiple pregnancies and suggest SET in
their infertility treatment programs.

Currently an ideal target is considered in ART;
a healthy baby is the success, not a positive pregnancy.
The majority of twin or high order multiple
pregnancies (HOMP) are not successful or associated
with poor maternal and neonatal outcomes
(13). De Neubourg and Gerris (14) have reported
that the twin rate after IVF/ICSI dropped by at
least 50% simply by transferring only one goodquality
embryo in the first and second fresh IVF/
ICSI cycles in young women, without a reduction
in the overall pregnancy rate. They believed that
preventing ‘the second half‘ of IVF/ICSI twins
constituted another, probably tougher challenge
because the target group was a heterogeneous
mix consisting of patients in very different clinical
situations. However, they suggested expanding the SET policy to women <38 years of age until
the third cycle and to cryoperservation cycles. In
many European countries the SET policy is the
primary prevention method used to prevent multiple
pregnancies. However, in Iran, the transfer of
just one embryo, even with excellent morphology
and quality, is taboo because it is feared that the
pregnancy rate will decline (15). Despite warnings
from physicians about the risks associated
with multiple pregnancies, patients that have longterm
infertility problems perceive twin or multiple
pregnancies as blessings from God. In some cases
they are not satisfied with multifetal pregnancy
reduction. However, physicians are ethically obligated
to inform the patients regarding the risks of
multiple pregnancies.

A limitation of the present study is its retrospective
nature; therefore, we could not evaluate the
effect of some possible factors reported in previous
studies.

Conclusion

We suggest the SET policy for prevention of
multiple pregnancies in women younger than 35
years of age who undergo ICSI cycles with high
fertilization rates and excellent or good quality
embryos. The remaining embryos should be cryopreserved.
However, further prospective studies
are necessary to evaluate whether SET in women
with these risk factors can significantly decrease
the multiple pregnancy rate and improve cycle
outcomes.

Acknowledgements

We would like to express our appreciation to our
co-workers and all participants involved in this
study. There is no financial support and conflict of
interest in this study.